1. Field of the Invention
The field of this invention is a method of targeting promoter-less selection cassettes into transcriptionally active loci, for the purpose of ensuring very high efficiency of targeting compared to traditional targeting methods.
2. Description of Related Art
Transgenic and knockout (KO) animals are used extensively to gain insight into gene function and to evaluate putative drug-targets in whole organisms. In the case of KO animals, a gene of interest is usually replaced by a marker gene to create a heterozygous null allele that can then be bred to homozygocity. In spite of the advantages and utility of transgenic animal technology, currently available methods for creating transgenic animals suffer from several technological problems. Some of the more serious problems associated with this method arise from the fact that DNA constructs introduced into genomes integrate randomly and frequently in multiple copies. In turn, this random integration can often lead to subsequent problems due to positional effects, silencing of the transgene, and insertional inactivation of an endogenous allele. A need still remains for methods that allow rapid, reproducible, efficient, and simple generation of transgenic and knockout animals that are devoid of the confounding issues that exist in currently available methods.
U.S. Pat. No. 6,150,169 describes methods for obtaining expression of a heterologous gene in a host. WO 99/53017 describes methods for generating transgenic animals. Vaulont et al. (1995) Transgenic Research 4:247-255 describes the use of a dicistronic promoter-less construct. Friedrich et al. (1991) Genes Development 5:1513-1523 describes random integration of a promoter-less vector into ES cells. Zambrowicz et al. (1997) Proc. Natl. Acad. Sci. USA 94:3789-3794 characterizes the ROSA26 locus.